DETECTION OF MENDELIAN AND GENOTYPE FREQUENCY OF GROWTH HORMONE GENE IN ONGOLE CROSSBRED CATTLE MATED BY THE ARTIFICIAL INSEMINATION TECHNIQUE U. Paputungan 1,2, L. Hakim3, G. Ciptadi3 and H.F.N. Lapian1 of Animal Science, Sam Ratulangi University, Manado 95115 - Indonesia 2Graduate School, Department of Animal Production, Brawijaya University,Malang 65145 - Indonesia 3Faculty of Animal Husbandry, Brawijaya University, Malang 65145 - Indonesia Corresponding E-mail: [email protected]

1Faculty

Received April 16, 2012; Accepted May 28, 2012

ABSTRAK Tujuan penelitian ini adalah menetapkan pewarisan gen hormon pertumbuhan (HP) melalui jenis Mendel dan menentukan frekuensi genotype HP pada sapi peranakan Ongole yang dikawinkan dengan teknik inseminasi buatan (IB). Sebanyak 76 sampel darah diambil dari induk Peranakan Ongole dan pejantan (G0), dan keturunannya (G1) di Desa Tumaratas sebagai Pusat Pelayanan IB provinsi Sulawesi Utara, Indonesia. Semua sampel darah diuji keberadaan lokus HP dengan memakai metode PCR-RFLP yang melibatkan restriksi enzyme Msp1 pada 1,2% gel agarosa. Data dianalisis dengan menggunakan fungsi program statistik pada Excel XP. Hasil menunjukkan bahwa lokus HP yang memakai alel restriksi enzim Msp1+ dan Msp1- dalam induk dan pejantan diwariskan pada keturunan Peranakan Ongole mengikuti pewarisan jenis Mendel. Pewarisan sifat jenis Mendel melalui teknik IB ini tidak berada dalam keseimbangan genetik untuk frekuensi genotipe Msp1 dalam kelompok G0 dan G1. Program pemuliaan dengan menggunakan variasi genotipe pejantan dan induk (G0) untuk mewariskan genotipe restriksi enzim Msp1 HP melalui teknik IB bisa dipertahankan untuk meningkatkan tingkat sebaran alel yang bervariasi guna meningkatkan keseimbangan genetik dan pemuliaan genotipe pada populasi sapi peranakan Ongole. Kata Kunci: frekuensi genotipe, Msp1 HP, sapi peranakan Ongole, pewarisan Mendel ABSTRACT The objectives of this study were to detect the Mendelian mode inheritance of growth hormone (GH) and to establish genotype frequency of GH gene in Ongole-crossbred cattle mated by the artificial insemination (AI) technique. Total of 76 blood samples were collected from Ongole-crossbred cows and bulls (G0), and their progenies (G1) at the Tumaratas AI service center in North Sulawesi province, Indonesia. All blood samples were screened for the presence of GH locus using a PCR-RFLP method involving restricted enzyme Msp1 on 1.2 % of agarose gel. Data were analyzed using statistical program function in Excel XP. The results showed that GH locus using alleles of Msp1+ and Msp1- enzyme restriction in Ongole-crossbred cows and bulls was inherited to their Ongole-crossbred progenies following the Mendelian mode inheritance. This Mendelian inheritance generated by AI technique was not under genetic equilibrium for the Msp1 genotype frequencies in groups of G0 and G1. The breeding program using genotypes of bulls and cows (G0) for generating the genotype of GH Msp1 enzyme restriction by AI technique should be maintained to increase these various allele dispersion rates for breeding under genetic equilibrium of the Ongole-crossbred cattle population. Keywords: genotype frequency, GH Msp1, Mendelian inheritance, Ongole-crossbred cattle

INTRODUCTION Growth hormone (GH) is an anabolic

hormone synthesized and secreted by the somatotroph cells of the anterior lobe of the pituitary in a circadian and pulsatile manner, the

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pattern of which plays an important role in postnatal longitudinal growth and development, tissue growth, lactation, reproduction, as well as protein, lipid and carbohydrate metabolism (Ayuk and Sheppard, 2006). GH gene with its functional and positional potential has been widely used for marker in several livestock species including the Indonesia local cattle (Jakaria et al., 2009; Sutarno, 2010). Ongole-crossbred cattle give a significant contribution to Indonesian national meat supplies to fulfill animal protein needs of people. However, the increase of cattle population is not balanced with the national needs of meat consumption due to higher increase of human population. If this condition is uncontrolled, it will lead to the loss of germplasms which is one of the national assets in the animal husbandry field. Negative selections by the breeders and a lack of observation for the crossbreeding of local cattle cause the rest of local cattle to be the inferior cattle with a low quality, will be used to serve as the parental animals in breeding program. If this conventional breeding happens continuously, it will lose the benefit due to the extinction of superior animal germplasms (Hardjosubroto, 2002). In the wide spread use of artificial insemination (AI) techniques in cattle reproduction industry, the Ongole bulls are used widely as sperm source in crossbreeding program to improve the performance of indigenous local breeds of the cattle in North Sulawesi province. As part of the marker assisted selection (MAS) program which aimed to improve genetic traits in bulls of the Ongole-crossebred cattle, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) has been identified at GH locus with Msp1 enzyme restriction. The uncontrol breeding of selected different genotypes of parental bull and cows by the AI technique could be the factor causing genetic inequilibrium of genotype frequency in animal population as a part of non random mating system (Van Vleck et al., 1987). The study of the Mendelian mode inheritance of GH Msp1 enzyme restriction has not been widely explored in cattle. The objectives of this study were to detect the Mendelian mode inheritance and to establish genotype frequency of growth hormone (GH) Msp1 enzyme restriction in Ongole-crossbred cattle mated by the artificial insemination (AI) technique in North Sulawesi, Indonesia.

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MATERIALS AND METHODS Animals and Sample Collection This study was carried out in North Sulawesi Province of Indonesia. The total of 74 female animals (parental cows and their progenies) were used and comprised of 37 cows (age ranging 4 to 5 years old), and their 37 female progenies of Ongole crossbred cattle (age ranging 35 to 56 days old). All cows were reared under private areas belong to farmers with unknown ancestors. Progenies were born from those cows mated by artificial insemination using germ plasms (semen) of the two Ongole bulls called “Krista” and “Tunggul” from the artificial insemination bull germ plasm center or Balai Besar Inseminasi Buatan (BBIB) in Singosari, East Java province, Indonesia. DNA Extraction The genotyping process was conducted at the Biotechnology Laboratory Department of Biological Science, Faculty of Mathematics and Natural Science, Sam Ratulangi University, Manado. Blood samples of the cows, their progenies and two Ongole bulls as source of germ plasms were collected from their Jugular vein in 10 ml EDTA (10%) tubes during July 2011 and stored in the refrigerator (4oC) until ready for DNA isolation. Genomic DNA from their whole blood were purified by standard protocol using proteinase K digestion as described by DNA extraction kit (AxyPrep Blood Genomic DNA Miniprep kit, AXYGEN Biosciences, Union city, CA, 94587, USA). Genotyping for GH and Allele Identification Following the genomic DNA isolation, the animals were genotyped for GH locus using PCRRFLP and 1.2% agarose gel electrophoresis (Sulandari and Zein, 2003). Amplification of the fragment of 327 bp at intron 3 (Gordon et al., 1983) was done with PCR using forward primer 5’-CCCACGGGCAAGAATGAGGC-3’; reverse primer 5’-TGAGGAACTGCAGGGGCCCA-3’ (Mitra et al., 1995). The reaction mixture of PCR was performed by using 1x Taq pol 25 µl of master mix (Axygen Biosciences, CA, USA). To digest this fragment, a protocol of RFLP with restriction enzyme Msp1 was used to recognize the particular site of C↓CGG. The PCR product of GH gene was digested at 37o C for 3 hours by Msp1 enzyme. Reaction consisted of 2

J.Indonesian Trop.Anim.Agric. 37(2) June 2012

µl Buffer V2 10X, 7.5 µL H2O, 0.5 µL Enzim Msp1 (20 U/µL), and 10 µl PCR product. PCR protocols to amplify the fragment were done by the initial denaturation temperature step at 94 oC for 5 min for 1 cycle followed by 35 cycles of denaturation at 94oC for 30 sec, annealing at 60oC for 30 sec, elongation at 72oC for 30 sec and a final extention at 72 oC for 1 minute (Dybus, 2002). Following the end of PCR and RFLP process, the products were then subsequently electrophorated using 1.2% agarose gel to identify polymorphism of allele based on the length of the band (Figure 1). Data Analysis PCR-RFLP data were used in establishing the observed homozygous Msp1+/+ genotype, heterozygous Msp1+/- genotype and homozygous Msp1-/- genotype. The Mendelian mode inheritance test of the observed homozygous Msp1+/+ genotypic, heterozygous Msp1+/genotypic and homozygous Msp1-/- genotypic distributions including the genetic equilibrium test of the observed Msp1 genotype frequency in animal population was calculated using Chisquare test (Byrkit, 1987) as follows: X2 



(f o  f e ) fe

2



f

f

2 o

N

e

Where X2=

is the Chi-square distribution, fo the observed frequency of the ijkth cell, and fe is the expected frequency of the ijkth cell. f e ijk cell 

 f x f f e i

e j

e ijk

f

is the total of observed frequency of the i th row;  f e  j is the total of observed frequency of the jth column; and  f is the total of observed frequency of the ijkth cell. Data were analyzed using software of the statistical program function in Excel XP (2007). e i

e ijk

RESULTS AND DISCUSSION Polymorphism Detection The Msp1 digestion of the PCR products produced the fragments of 104 bp and 223 bp for allele Msp1+ and of 327 bp for allele Msp1(Figure 1). These alleles were the same as research reported by Zhou et al. (2005) using

Msp1 enzyme restriction in Beijing Holstein and Maylinda (2011) using Msp1 enzyme restriction in Grati dairy cows. This enzyme recognized only the restriction site of four nucleotides for C↓CGG (Figure 2). Genotype Msp1+/+ consisted of two bands (104 bp, 223 bp), genotype Msp1+/consisted of three bands (104 bp, 223 bp, 327 bp), genotype Msp1-/- consisted of one band (327 bp). The difference of these two fragments of Msp1+ and Msp1- alleles was caused by mutation of Cytosine (C) to Thymine (T) (Rifa ‘i, 2010). Gene variation of GH locus for Msp1 in cattle was detected in the position of intron 3 (Rifa’i, 2010) at the sequence position of 1547 based on nucleotide sequence from GenBank, number: M57764.1 (http://www.ncbi.nlm.nih.gov) sourced in Gordon et al. (1983) accessed on March 26, 2011. Mutation occurred on DNA level due to nucleotide changes, either transition or insertion (Cambell and Reece, 2008). Based on the difference of nucleotide restriction sites of each allele, the mutation of Cytosine (C) into Thymine (T) occurred due to nucleotide transition (Figure 2). The transition of C into T changed the restriction site of Msp1 enzyme (Rifa’i, 2010). Mendelian Mode Inheritance In this study, matings of the 14 homozygous genotyped parental cows of Msp1-/- with homozygous genotyped bull of Tu_-/- produced the all 14 homozygous genotyped progenies of Msp1-/-. In addition, matings of the 4 homozygous genotyped parental cows of Msp1-/with homozygous genotyped bull of Kr_+/+ produced the all 4 heterozygous genotyped progenies of Msp1+/- (Table 1). The same patterns were also observed that matings of the 3 homozygous genotyped parental cows of Msp1+/ + with homozygous genotyped bull of Kr_+/+ produced the all 3 homozygous genotyped progenies of Msp1+/+. In the same observation, matings of the 2 homozygous genotyped parental cows of Msp1+/+ with homozygous genotyped bull of Tu_-/- produced the all 2 heterozygous genotyped progenies of Msp1+/- (Table 1). Theorically, the basic Mendelian mode inheritance of the crossing between all the same homozygous genotyped individuals produced all the same homozygous genotyped progenies, while the crossing between recessive homozygous genotyped individuals and dominant homozygous genotyped individuals produced all heterozygous genotyped progenies (Van Vleck, 1987). Based on the Chi square test (Table 1), it was found that the

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Figure 1. Genotyping Results of Msp1 Enzyme Restriction in GH Locus Detected by Agarose Gel Electrophoresis

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J.Indonesian Trop.Anim.Agric. 37(2) June 2012

Figure 2. Fragment difference of GH gene and restriction site of Msp1 enzyme based on GH gene sequence in cattle accessed from GenBank, number: M57764.1 (http://www.ncbi.nlm.nih.gov).

Table 1. Distribution of Parental and Progeny Genotypes of Msp1+/+ and Msp1–/– Enzyme Restriction at Growth Hormone (GH) Locus in Ongole crossbred cattle in North Sulawesi Based on Genotyping Results Detected by Agarose Gel Electrophoresis Parental Cow Genotype Msp1-/-

n

14 4

Parental Bull Genotype used in the AI technique Tu_ Msp1-/Kr_ Msp1+/+ Sub total

Msp1+/-

Msp1+/+

Genotype of Progeny (F1) -/-

+/-

+/+

Total

Obs

Exp

Obs

Exp

Obs

Exp

14

14

0

0

0

0

0

0

4

4

0

0

4 2

0 NS 0

0

3

4 NS 3

14NS 2

14

18 NS

0

5

Tu_ Msp1-/-

9

Kr_ Msp1+/+

0

0

6

5

3

4

Sub total

2 NS

3

9 NS

7

3 NS

4

2

Tu_ Msp1-/-

0

0

2

2

0

0

3

Kr_ Msp1+/+

0

0

0

0

3

3

Sub total

0 NS

0

2 NS

2

3 NS

3

5 NS

16 NS

17

15 NS

13

6 NS

7

37 NS

Total

14 NS

n = number of parental cows mated by bull using the artificial insemination (AI) technique. Tu = Tunggul (name of Ongole bull), Kr = Krista (name of Ongole bull). Obs = Observed; Exp = Expected. NS) X2=1.15 < X2 {2}=5.991; the values denoting progeny genotypic distributions were under Mendelian mode 0.05 inheritance (P X2 {2}=5.991; the values denote that genotype frequencies of the parental cows and 0.05 their progeny populations were not in genetic equilibrium (P>0.05) based on the Chi square test.

progeny genotypic distributions were under the Mendelian mode inheritance as denoted by the value of X2=1.15 < X20.05{2}= 5.991. The Msp1 Genotype Frequencies of Animal Population Using AI Technique The frequencies of cow (G0) and progeny (G1) genotypes determined in the population mated by each genotype of bull (G0) were presented in Table 2. Based on the Chi Square test (Table 2), it was found that genotype and allele frequencies of GH Msp1 were not under genetic equilibrium (P>0.05). Maylinda (2011) reported that Grati dairy cow population was in genetic equilibrium. This was supported by the fact that a population property of gene pool for GH-Msp1 under the Hardy-Weinberg equilibrium pattern was a function of both allele frequencies and biological interactions among genes (Carter et al., 2005). This inequilibrium of genotypic frequencies of GH Msp1 caused the instability of genotypic frequencies of GH gene from G0 generation to the next generation (G1) due to the breeding of selected genotypic bulls and parental cows without random mating system in animal population (Cambell and Reece, 2008; Rifa’i, 2010). The factor affecting genetic equilibrium was selection program with non random mating system, such as the artificial insemination mating

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system (Van Vleck et al., 1987). Jawasreh et al. (2012) reported that breeding program must be continued as the first step to increase the frequency of the favorable allele in breeding station. In North Sulawesi province, the artificial insemination service center applied the straw containing spermatozoa germplasm of Ongole bull called “Krista” and “Tunggul” from germplasm center (Balai Besar Inseminasi Buatan) Singosari, East Java province. Carter et al. (2005) reported the analysis of gene interaction and found that it might be two or more genes can interact to express a particular phenotype. CONCLUSION The selected growth hormone locus using alleles of Msp1+ and Msp1- enzyme restriction in Ongole-crossbred parental cows and bulls was inherited to their progenies following Mendelian mode inheritance. This Mendelian inheritance generated by AI technique was not under genetic equilibrium for Msp1 genotype frequencies in groups of parental animals (G0) and their progenies (G1). The breeding program using genotypes of bulls and cows (G0) for generating the genotype of GH Msp1 enzyme restriction by AI technique should be maintained to increase these various allele dispersion rates for breeding under genetic equilibrium of the Ongole-

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crossbred cattle population. ACKNOWLEDGMENT The financial support of the Ministry of Education and Culture, Republic of Indonesia through their Research under Doctorate Scholarship Program is gratefully acknowledged. The authors also acknowledge J. Kuhu and his farmer group members at Tumaratas village, district of West Langowan, under development of the artificial insemination service center of Minahasa regency, North Sulawesi province for their assistance in animal data collection. REFERENCES Ayuk, J. and M.C. Sheppard. 2006. Growth hormone and its disorder. Postgrad. Med. J. 82(963):24-30 Byrkit, D.R. 1987. Statistics Today: A Comprehensive Introduction. The Benjamin/Cummings Publishing Company, Inc. 2727 Sand Hill Road Menlo Park, California, USA. Cambell, N.A. and J.B. Reece. 2008. Biology. Eighth Edition. Pearson Education Inc., California. Carter, A.J.R., J. Hermisson and T.F. Hansen. 2005. The role of epistatic gene interactions in the response to selection and the evolution of evolvability. Theor. Popul. Biol. 68:179196 Dybus, A. 2002. Associations between Leu/Val polymorphism of growth hormone gene and milk production traits in Black-and-white cattle. Archives Anim. Breeding. 45(5):421428 Gordon, D.F., D.P. Quick, C.R. Ewin, J.E. Donelson and R.R. Maurer. 1983. Nucleotide sequences of the bovine growth hormone chromosomal gene. GenBank, number: M57764.1 (http://www.ncbi.nlm.nih.gov) accessed on March 26, 2011 Hardjosubroto, W. 2002. Arah dan sasaran penelitian dan pengembangan sapi potong di

Indonesia: Tinjauan dari segi pemuliaan ternak. Workshop sapi potong, Malang, 1112 April 2002. Jakaria, R.R. Noor, H. Martojo, D. Duryadi and B. Tappa. 2009. Identification of growth hormone (Gh) gene MspI and AluI loci polymorphism in beef cattle. Faculty of Animal Science, Bogor Agricultural University. Proceedings, the 1st International Seminar on Animal Industry 2009. p.42-47. Jawasreh, K.I.Z., H. Ababneh, F.T. Awawdeh, M.A. AL-Massad and A.M. Al-Majali. 2012. Genotype and allelic frequencies of a newly identified mutation causing blindness in Jordanian Awassi sheep flocks. Asian-Aust. J. Anim. Sci. 25(1):33-36. Maylinda, S. 2011. Genetic polymorphism of growth hormone locus and its association with body weight in Grati dairy cows. International J. Biotechnology and Moleculer Biology Research. 2 (7):117-120 Mitra, A., P. Sciilee, C.R. Balakrisiinan and F. Pirciiner. 1995. Polymorphisms at growth hormone and prolactine loci in Indian cattle and buffalo. J. Anim. Breed. and Genet. 12:71-74 Rifa’i, M. 2010. Genetika Rekombinasi dan Populasi. Edisi Pertama. Penerbit Galaxy Science, Malang, 65145 Sulandari, S. and M.S.A. Zein. 2003. Protocols in DNA Laboratory, Center of Biology Research, The Indonesian Institute of Sciences, Bogor. Pp.23-45 Sutarno. 2010. Genetic variation among Indonesian native cattle breeds based on polymorphisms analysis in the growth hormone loci and mitochondrial DNA. Biodiversitas 11:1-5 Van Vleck, L.D., E.J. Pollak and E.A.B. Oltnacu. 1987. Genetics for the Animal Science. W.H. Freeman and Company, New York. Zhou, G.L., H. Liu, C. Liu, S.L. Guo, Q. Zhu and Y.H. Wu. 2005. Association of genetic polymorphism in GH gene with milk production traits in Beijing Holstein cows. J. Biosci. 30(5):595-598

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